The goal of this project is to understand the role of histone macroH2A proteins in chromatin structure and function. There are three macroH2A subtypes that are encoded by two macroH2A genes, macroH2A1 and macroH2A2. All three subtypes have a similar unusual structure that includes an H2A domain and a large nonhistone domain. MacroH2A proteins are evolutionarily highly conserved among vertebrates and are present in many mammalian cell types where they replace conventional H2A in a subset of nucleosomes. MacroH2A proteins are preferentially associated with the inactive X chromosome of female mammals, suggesting that they have a role in silencing transcription through chromatin structure. MacroH2As may also be involved in regulating gene expression in autosomal regions. Understanding the function of macroH2A proteins may give insights into human disorders, such as cancer, that involve disruptions of normal gene regulation. A better understanding of chromatin mediated gene repression could also be useful for gene therapy, where potentially therapeutic genes are often inactivated after introduction into target cells. Our experimental approach will focus on the use of mice carrying knockout mutations in the macroH2A1 and macroH2A2 genes. These mice, and organs and cells prepared from them, will be examined for specific phenotypic and pathological effects related to the absence of macroH2A proteins. We will examine the effects of the knockout mutations on X-inactivation and the expression of genes from other chromosomes. The distribution of macroH2A proteins in the chromatin of normal mice will be examined by purifying macroH2A-containing chromatin fragments. The DNA associated with these fragments will be used to identify regions of the genome where macroH2A proteins are concentrated, and these regions will be examined for the effects of macroH2A proteins on chromatin structure. We will also examine the protein composition of the macroH2A-containing chromatin fragments in order to identify proteins that interact with macroH2A-containing chromatin and therefore, may be involved in its function in the chromatin.